Intra‐articular injection of rapamycin microparticles prevent senescence and effectively treat osteoarthritis

Abstract Trauma to the knee joint is associated with significant cartilage degeneration and erosion of subchondral bone, which eventually leads to osteoarthritis (OA), resulting in substantial morbidity and healthcare burden. With no disease‐modifying drugs in clinics, the current standard of care focuses on symptomatic relief and viscosupplementation. Modulation of autophagy and targeting senescence pathways are emerging as potential treatment strategies. Rapamycin has shown promise in OA disease amelioration by autophagy upregulation, yet its clinical use is hindered by difficulties in achieving therapeutic concentrations, necessitating multiple weekly injections. Rapamycin‐loaded in poly(lactic‐co‐glycolic acid) microparticles (RMPs) induced autophagy, prevented senescence, and sustained sulphated glycosaminoglycans production in primary human articular chondrocytes from OA patients. RMPs were potent, nontoxic, and exhibited high retention time (up to 35 days) in mice joints. Intra‐articular delivery of RMPs effectively mitigated cartilage damage and inflammation in surgery‐induced OA when administered as a prophylactic or therapeutic regimen. Together, the study demonstrates the feasibility of using RMPs as a potential clinically translatable therapy to prevent the progression of post‐traumatic OA.

upregulation during these stress conditions can make the cells apoptotic, leading to diminished repair and remodeling capability in cartilage. 12,13,[17][18][19] Chondrocytes under constant oxidative or genotoxic stress conditions can also turn senescent, leading to the secretion of inflammatory cytokines. [20][21][22] The senescent chondrocytes secrete a host of inflammatory proteins such as IL-6, IL-8, and IL-17, together known as senescent-associated secretory phenotype (SASP) factors, which attracts immune cells leading to chronic inflammation. 23,24 Selective modulation of senescent cells has been one of the widely sought-after OA disease-modifying strategies, where a wide variety of senolytic or senomorphic drugs are being evaluated, with a few of them undergoing clinical trials. [25][26][27][28][29] Together, these studies suggest that autophagy activation and senescence modulation are critical for maintaining homeostasis of the articular cartilage, and targeting these mechanisms can act as novel treatments to modify OA outcomes. 26,27,[29][30][31][32] Rapamycin, a well-known immune modulator and an antibiotic used routinely in clinics for various diseases, has been shown to delay OA progression in mice models. 30,33 Rapamycin induces autophagy via mTOR inhibition, pushes the cells from stress into the nutrient survival pathway, and restores the chondrocyte's health. 30,33,34 Rapamycin also prevents senescence and stalls the production of SASP factors. [35][36][37][38][39] It is hence evident that rapamycin is a promising drug for OA treatment. However, free drug administration via intraarticular (IA) injection is challenging due to effective lymphatic clearance in joints and hence necessitates high dose administration and frequent injections. Compounds with a molecular weight close to rapamycin (914 g mol À1 ) such as Evans blue (963 g mol À1 ) and Ceftazidime (564 g mol À1 ) exhibit IA residence time of less than 1 h. 40 Small molecules (<10 kDa) such as corticosteroids frequently administered intra-articularly for pain management of OA have residence time only up to 1-4 h. 41 Repeated injections to sustain therapeutic effect can lead to pain, joint irritations and infections, and poor clinical acceptance.
Studies that have attempted to deliver OA disease-modifying agents such as inhibitors of catabolic enzymes and cytokine receptors via IA injections have met with little clinical success. [42][43][44] One of the main reasons for the lack of success of these potential diseasemodifying agents is an insufficient therapeutic concentration for prolonged duration in the joint, owing to the efficient and rapid lymphatic clearance inside the knee joint. 41 Senolytic drug, UBX0101, which was promising in treating OA in mice, 25 recently failed to show efficacy after 12 weeks of a single IA injection (maximum dose of 4 mg per patient) in phase II clinical trial (NCT04129944). It is hypothesized that the lack of efficacy of UBX0101 was due to its rapid clearance from the joints. Another phase I clinical trial using two injections (4 mg each) at weeks 0 and 4 is underway (NCT04229225). Thus, it becomes evident that sufficient and prolonged therapeutic concentration inside the articular joints necessitates several frequent administrations, which significantly reduces patient compliance at clinics. IA drug delivery systems using polymer-based slow-release formulation can serve as the key in bringing many potent drugs into clinics. 45,46 Poly lactic-co-glycolic acid-based system (PLGA) is a robust and widely used clinical drug delivery system. 46,47 We previously showed that PLGA-based drug delivery systems have prolonged retention time in the murine knee joint. 37 Therefore, we hypothesized that IA injections of rapamycin in PLGA-based microparticles (RMPs) could prolong the drug's residence time and could be used to treat OA.
We report that the rapamycin PLGA microparticles induce autophagy and prevent senescence in primary human articular chondrocytes (HACs) obtained from OA patients. The formulation sustained the production of sGAG production in stressed micromass cultures. When administered as prophylactic or therapeutic regimens, the rapamycin particle formulation ameliorates surgery-induced OA in mice. To our knowledge, this is the first report of successful mice OA therapy using rapamycin in an injectable microparticle formulation, and such strategies should be explored further for translation to humans. which is in line with previously published results. 13,48,49 2.2 | PLGA microparticles provide a tunable platform to release rapamycin Size plays a vital role in the prolonged joint retention time of PLGA microparticles. We had previously shown that particles in a size range of 1 μm exhibited high residence time in murine 37 and rodent 50 knee joints, and hence we proceeded to use this size range for our current study. Dynamic light scattering analysis revealed an average diameter of 1039 ± 188 nm ( Figure 2a; Table S1), and the zeta potential was À18 mV due to the presence of acid end-capped PLGA polymer in the particles (Figure 2b).
To assess the tunability, microparticles of different molecular weights of PLGA were used to encapsulate rapamycin. The size and rapamycin encapsulation efficiency (EE) are listed in Supplementary - Table S1. It was observed that the PLGA MPs of molecular weight

| RMPs induce autophagy in primary HACs
Rapamycin is a well-known autophagy inducer that acts by inhibiting the mTOR signaling pathway. 51 We had previously shown that rapamycin as a free drug and MP formulation induced autophagy in the C28/I2 (human chondrocyte cell line) ( Figure S2a-e). 37 Here, we found that in the primary HACs, both free rapamycin and RMPs successfully induced autophagy as visualized by the LC3B puncta in the cells.

| RMPs prevented senescence in primary HACs
Chondrocytes in the knee joint get exposed to various stress conditions such as DNA damage and increased ROS production. 52 Figure S3a). Next, we

| RMPs help to sustain sGAG production in stressed micromass cultures
When seeded at a high density along with the growth factor (TGF-β), chondrocytes form three-dimensional (3D) micromasses that exhibit high deposition of extracellular matrix components such as sulfated glycosaminoglycans (sGAG). 55 They serve as in vitro 3D culture models and are widely used in cartilage research to evaluate various stress conditions and treatment modalities. The micromasses derived from C28/I2 cells were treated with genotoxic or oxidative stress to simulate physiological stress conditions present during knee joint trauma. These were also cotreated with rapamycin as a free drug or MP formulation to evaluate the sGAG production in the stressed micromasses.
The concentrations of BrdU (600 μM) and H 2 O 2 (100 μM) were obtained by our initial optimization experiments. In the BrdU optimization experiment, two doses of BrdU were chosen: 400 and 600 μM.
The higher dose of BrdU (600 μM) reduced the sGAG production by twofold compared to untreated, and hence we continued to use this The micromasses derived from HACs were treated with oxidative stress using externally added H 2 O 2 (100 μM) and cotreated with free rapamycin or RMPs. We wanted to evaluate whether rapamycin or RMPs can sustain the production of sGAG in micromasses under oxidative stress conditions. In micromasses derived from HACs, H 2 O 2 treatment resulted in nearly threefold lower sGAG production than the vehicle-treated groups, while the free rapamycin or RMPs treatment rescued the sGAG production and was comparable to untreated groups ( Figure 3f). Similar results were also obtained with micromasses formed from C28/I2 cells when treated with oxidative (H 2 O 2 , 100 μM) or genotoxic (BrdU, 600 μM) stress for 48 h ( Figure S7a,b).
To evaluate the long-term sGAG production by the stressed micromasses, we treated micromass formed from C28/I2 cells for 8 days with BrdU (600 μM) or H 2 O 2 (100 μM) along with cotreatment groups containing rapamycin or RMPs at 1 μM dose. In the BrdU (600 μM) treated group, the absorbance values dropped to almost sixfold compared to untreated groups, while the RMP-treated groups sustained the sGAG production on par with free rapamycin-treated groups ( Figure S7b). Likewise, sGAG production dropped to almost threefold with H 2 O 2 treatment, and the rapamycin or RMP-treated group significantly increased the sGAG production at par with F I G U R E 3 RMPs prevented senescence in primary HACs and sustained sGAG production in micromass cultures exposed to oxidative stress. SA-β Gal-stained images of primary HACs exposed to (a) no treatment, (b) oxidative (H 2 O 2 ) stress, (c) oxidative (H 2 O 2 ) stress with free rapamycin (1 μM), and (d) oxidative (H 2 O 2 ) stress with RMPs (1 μM). Scale bar -40 μm. (e) Percent of senescent HACs after culturing in oxidative (H 2 O 2 ) stress condition and different cotreatments (n = 3 per group) for 48 h. (f) sGAG production in stressed micromass cultures measured as absorbance of guanidine HCl extracted Alcian blue stain. Images and graphs were representatives of data collected from three osteoarthritis (OA) patients. Data in graphs represent the mean ± SD and p values were determined by one-way analysis of variance (ANOVA) and Tukey's post hoc tests. p-value <0.05 was considered significant. BMPs, blank microparticles; HACs, human articular chondrocytes; ns, nonsignificant; RMP, rapamycin-loaded microparticles. ****p < 0.0001 untreated groups ( Figure S7d). In summary, these results suggest that rapamycin and RMPs were potent anabolic agents and sustained sGAG production in stressed chondrocytes for long durations under oxidative and genotoxic stress.

| PLGA MPs of high molecular weight had longer residence time in mice knee joints
To estimate the dosage and frequency of rapamycin to be administered for mice models of OA, we wanted to determine the residence time of rapamycin in the knee joint space. Since in our in vitro rapamycin release data, 75-85 kDa PLGA MPs showed sustained release for more than a month (Figure 2c), we chose this formulation for all subsequent mice experiments. As it is difficult to assess the residence time of nonfluorescent drugs like rapamycin, we assessed the in vitro release rate of a fluorescent molecule, Cy7, which has a comparable molecular weight (Mw) (Mw of Cy7: 626.7 g mol À1 ; Rapamycin: 914.1 g mol À1 ) and hydrophobicity (LogP of Cy7 3.4; LogP of Rapamycin: 4.81) as rapamycin. The in vitro release profile of the RMPs and the Cy7 particles in 1x phosphate-buffered saline (PBS; Figure S8) followed a similar trend, and a two-phase decay curve using nonlinear regression (least square method) was used to fit the release pattern of rapamycin and Cy7 dye from PLGA particles. Rapamycin and Cy7 dye followed a typical biphasic release from PLGA MPs, in line with the previously published literature. 37,56,57 Next, we injected Cy7 containing PLGA MPs intra-articularly in mice and monitored the residence time inside the knee joints using an in vivo imaging system -Perkin Elmer IVIS ® Spectrum. The contralateral legs of mice received an equal concentration of Cy7 free dye ( Figure S9). The joints receiving free dye injections had 7% of the initial injected dose remaining on day 3, whereas the Cy7 MPs injected group showed fluorescent signal even on day 35 ( Figure S9c). The loss of injected free dye could be attributed to the clearance by the lymphatics in the joints or degradation and loss of fluorescence. The joints did not show any gross signs of inflammation, infections, or difficulty in movements during the entire course of this study. These results align with our previously published work, indicating that dyes encapsulated in 1 μm particles exhibit higher residence time inside mice and rodent knee joints than free dyes. 37   Other promising OA drugs can also be codelivered using this platform to explore synergy in OA treatment.
Drugs that exhibit multifaceted approaches such as autophagy activation, senescence prevention, and reducing inflammation in the milieu can potentially turn into promising therapies that are translatable. 72,73 Autophagy is a stress survival mechanism that gets activated as an adaptive process to different forms of metabolic stress, and the role of autophagy in OA is widely studied. 8,12,15 Cellular senescence is another growing field and is an important target for many diseases, including OA. 48,[74][75][76] In the mice model of OA, fewer cells were positive for LC3B (Figures 4 and 5e,f), and a higher number of cells were positive for the senescence marker -p16 INK4a (Figures 4 and 5g,h) compared to the sham surgery group. In contrast, the RMP-treated groups upregulated autophagy (Figures 4 and 5e,f)  OA. Since OA is predominantly a geriatric disease, and aged mice report a higher senescence burden and develop more severe OA after injury, additional evaluation into aged mice and genetically modified OA models, such as STR/Ort mice, can broaden the scope of our formulation. 83,84 Preclinical investigations are also necessary in larger animal models that may require additional optimization of dosages and treatment regimens before translation to humans.
In summary, we report the development of rapamycin as an MP formulation that can be administered after articular joint injury to prevent OA. This formulation was potent to prevent senescence and    Table S2.

| Synthesis of PLGA microparticles
PLGA microparticles were synthesized from PLGA polymer of various molecular weights, using the single emulsion technique described earlier. 37 Briefly, 100 mg of PLGA was dissolved in 2 mL of dichloromethane (DCM) with or without rapamycin (1 mg), and the homogenization was carried out in 1% polyvinyl alcohol (10 mL) at 12,000 rpm.
This solution was added to 1% PVA (110 mL) and was allowed to stir continuously for 3-4 h to evaporate DCM completely. The solution was then centrifuged at 11,000 Â g, and the pellet was washed using deionized water twice to wash away the excess PVA. The microparticles were then resuspended in deionized water and rapidly frozen at   were taken per well. These images were analyzed using ImageJ software.
To automatically score the senescent cells, we developed a custom-built macros algorithm to score the senescent cells based on their size and the intensity of SA-β Gal staining. Bright-field images were taken from each treatment group (vehicle-1x PBS for H 2 O 2treated group and DMSO for BrdU-treated group). The macros algorithm was run to count the total number of senescent cells.

| RMPs treatment in senescence induction assays (micromass culture)
We generated micromasses as described previously. 55 Briefly, the cells were seeded as a 15 μL suspension in growth media in a 24-well plate at a density of 2.5 Â 10 7 cells mL À1 . The cells were allowed to adhere to the well plate for 3 h, after which growth media was added.

| Immunocytochemistry
After 48 h of treatment, the cells were fixed using 4% PFA followed by permeabilization using 0.05% Triton X-100. The cells were then blocked using 5% nonfat dry milk and incubated overnight at 4 C with rabbit anti-LC3B polyclonal antibody (0.5 μg mL À1

| Effect of RMPs on surgically induced OA
Male WT C57BL/6 mice (25 g) were used for this study. 86 Following intraperitoneal injection of ketamine (80 mg kg À1 ) and xylazine (10 mg kg À1 ) as a combination in sterile 1x PBS, the animals were rested until the surgical plane was reached with no active plantar reflexes.
DMM was performed in mice knee joints to induce post-traumatic OA. A medial parapatellar incision exposed the knee joint capsule. The patella was displaced laterally after the joint capsule was opened, followed by transection of the medial meniscotibial ligament using surgical blade number 11 and scalpel. Following irrigation of the operated site with saline, the capsule and skin were sutured separately using absorbable PGA sutures. Sham groups received only an incision to expose the joint capsule, then sutured back like the other DMM-operated groups.
The mice were not immobilized and could move freely in the cage postoperatively. Each experimental group was evaluated by gross morphological examination for swelling, pain, or change in gait of the animal postsurgery and during the entire duration of the experiment.

| Classification of study
Prophylactic dose study: Group 1: DMM operated animals with no treatment (4 mice).
Group 2: Surgical control receiving no treatment (Sham) (4 mice). In the prophylactic study, IA injection was given 7 days after DMM surgery, followed by injections at days 24 and 42 with euthanasia at day 60. In the therapeutic study, the first injection was given on day 24 after DMM surgery, followed by a second injection on day 42 with euthanasia at day 60.

| Histology
The joints of operated knees were fixed in 4% paraformaldehyde for 24 h. The joints were embedded in paraffin after decalcification in 5% formic acid for 5 days. Five-micron sections were stained with Safranin-O-fast green staining using the described protocol. 89 In brief, slides were deparaffinized with xylene and alcohol. After a 5-min wash in running tap water, slides were stained with fresh Wiegert's iron hematoxylin for 10 min. Following a 5-min wash in running tap water, slides were soaked in 0.1% Safranin-O (10 min) and 0.05% fast green (5 min) in this order. After dehydration with alcohol and xylene, the slides were mounted with coverslips using mounting media. The medial tibial plateaus were imaged in the prepared slides and graded according to the OARSI OA cartilage histopathology assessment system. 90 Veterinarians performed the scoring in a blinded manner to minimize observer bias.
Similar steps were followed for hematoxylin and eosin staining as described above until Wiegert's iron hematoxylin stain. Following a 5-min wash in running tap water, slides were soaked in 1% eosin staining solution for 5 min followed by rinsing under tap water for 5 min. After dehydration with alcohol and xylene, the slides were coverslipped using mounting media, and the synovial lining of the different treatment groups was imaged.

| Immunohistochemical studies
Sections were processed similar to Safranin-O staining until the alcohol step followed by heat-induced epitope retrieval at 95 C in 1x TBST buffer (pH 9), followed by blocking using nonfat dry milk (0.8%).
The slides were rinsed in 1x TBST and incubated with MMP-13, ADAMTS-5, LC3B, and p16 INK4a antibodies (1:300) diluted in blocking solution at 4 C for 16-18 h. The slides were again washed in 1x TBST followed by incubation with HRP conjugated secondary antibody (1:150) diluted in blocking solution at room temperature for 2 h. The slides were again rinsed, and DAB (3,3 0 -diaminobenzidine) substrate was added and incubated for 1 h. Before brightfield imaging, the slides were washed, dried, and coverslipped using mounting media. The number of cells positive in each image was quantified using an automated macros program in ImageJ software.

| Statistical analysis
All statistical analysis was performed using Graph Pad Prism 6. Data were presented as mean ± SD. Nonlinear regression (least square method) two-phase exponential decay curve was used to fit rapamycin release profile with constrains of maximum value as 100 and minimum value as 0. Differences between groups were analyzed by t test or one-way analysis of variance (ANOVA), and nonparametric data were analyzed using the Mann-Whitney U test or Kruskal-Wallis test with Tukey's/Dunn's multiple comparison test, with p < 0.05 considered significant. Since the mean and SD estimate for each experimental animal group was not available initially, we kept a minimum of four animals in each group. After the results were available, we retrospectively calculated the statistical power to detect differences between free rapamycin and RMPs treatment groups using G*Power 3.1 software. The power for both prophylactic and therapeutic mice OA studies was greater than or equal to 80%.

| Data availability
All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).

| Code Availability
The macros code used for analysis during the current study are available from the corresponding author on reasonable request. Agarwal: Conceptualization (lead); investigation (supporting); project administration (lead); resources (lead); software (supporting); supervision (lead); validation (supporting); visualization (supporting); writingreview and editing (lead).

CONFLICT OF INTERESTS
The other authors declare that they have no competing interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.